This application is the national filing of PCT application No. PCT/GB91/00308.
It is common practice in the fields of biomedicine and molecular biology to use techniques for the analysis of biological molecules where the analytes are immobilised on the surface of membrane filters. Such techniques are well documented in the scientific literature and have been used for the analysis of DNA, RNA and protein molecules. The processes used in such analyses are commonly termed Southern blotting (1) where DNA molecules or fragments are analysed, Northern blotting (2) where RNA molecules or fragments are analysed and Western blotting (3) where proteins or polypeptides are analysed.
The principle of all these techniques involves separation of the analyte molecules by electrophoresis in agarose or acrylamide gels such that the molecules or fragments of molecules are separated according to their molecular weight or physical size. Separation is followed by transfer of the analyte to a membrane composed of natural or synthetic polymer to produce an exact replica of the separation pattern on the gel. Such transfer is commonly achieved by capillary transfer, application of an electric field or by use of vacuum suction.
In addition to the above techniques there are a variety of further blotting procedures in common use in biomedicine and molecular biology. The technique commonly known as the unblot (4) process involves drying the gel used for separation of the analyte to form a thin sheet which can then be handled and treated identically to a membrane blot. The techniques known as dot blotting (5) and slot blotting (6) are also widely used to confirm the presence or absence of a particular molecule or molecular fragment in an unseparated analyte mixture. In these techniques the analyte is applied directly to the membrane filter, either by use of a pipette tip, or by using a manifold filtration device.
Further blotting techniques are used in the process of gene cloning, to identify organisms containing an inserted nucleic acid sequence. In these procedures, known as colony lifts where bacteria are the organism used, or plaque lifts where the organism is viral, a filter is laid on the surface of a culture plate containing the organism and then removed, forming a replica of the distribution of organisms on the culture plate, which is then analysed for the presence or absence of the inserted nucleic acid sequence.
Central to all of the above techniques is the requirement to expose the immobilised analyte to a solution containing a further biomolecule, commonly termed a probe, which has an affinity for one or more analyte molecules, such that the probe molecule is able to bind to the immobilised analyte.
The probe molecule, which may be DNA, RNA, a synthetic oligonucleotide, a protein or polypeptide, or a ligand, is labelled with a radioactive isotope or non-radioactive reporter molecule, which may be either an enzyme or a small molecule (typically biotin or a hapten) recognised by an antibody or other binding protein.
Following exposure to the probe the blot is washed in order to achieve removal of unbound probe. Where the probe is labelled with a radioisotope, exposure of the blot to X-ray film produces an image on the film corresponding to the location of the analyte molecule or molecules recognised by the probe.
Where the probe carries a non-isotopic label further stages are necessary to achieve localisation of the bound probe. In the case of enzyme labelled probes, addition of a specialised enzyme substrate solution leads to the formation of a coloured stain or the emission of light at the position of the probe. Where the probe is labelled with biotin or a hapten molecule the blot is exposed to a solution containing either avidin or steptavidin in the case of biotin labelled probes, or to a solution containing an antibody recognising the hapten molecule. The antibodies or biotin binding proteins used in these procedures may themselves be labelled with either a radioisotope or an enzyme label, and detection is achieved using X-ray film or an enzyme substrate as described above.
While the great number of probe detection systems vary widely in method of action and in the complexity of the techniques used, all systems share a common requirement for multiple exposure of the immobilised analyte to reagent solutions, with each exposure separated by a washing procedure to prevent carry over or to remove non-specific binding of reagent, and to prevent interference with the next stage in the detection process.
Conventional methods of processing membrane blots to detect analytes are well established and documented (7,8,9,10). All methods rely on using a container or chamber in which the blot is placed, and into which the necessary reagent solutions may be introduced, in order to effect the desired detection of the analyte.
Such containers or chambers are typically plastic bags, plastic boxes, or plastic or glass tubes, or various combinations of containers may be used in a single procedure. The use of such containers is well documented in the scientific literature, and a variety of commercially produced apparatus are available.
Currently available methods all rely on varying degrees of manual intervention during the process of probing and detection of probe, and require the worker to carry out manual addition and removal of the various reagents used. As a consequence, the techniques used are labour intensive, time consuming and unsuitable for automation due to the intricacy of the manipulations involved in transfer of membranes in and out of containers, and in addition and removal of reagents without causing damage to the blot.
The invention as described seeks to remove the need for manual handling of the blot during processing and to perform the process of detection of analyte automatically by introduction of all necessary reagent and washing solutions onto the blot. Furthermore, by inclusion of a blot feed mechanism the invention may be used to allow the automatic sequential processing of a number of blots according to programmed instructions.